Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.15015/00001197

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Other Link： http://id.nii.ac.jp/1246/00001197/

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

Aquaporins facilitate water flux across biomembranes in cells and are involved in various physiological phenomena in several plant tissues. Generally, the water-flux activity of exogenously expressed aquaporins is measured in Xenopus laevis oocytes or yeast. However, heterogeneous systems are not likely to be optimal for plant aquaporin analysis. Thus, we created a new experimental system for functional analysis of plant aquaporins using lily (Lilium longiflorum) pollen protoplasts. Large protoplasts with uniform diameters of approximately 95m m were isolated from lily pollen grains. No plasma membrane intrinsic protein (PIP) aquaporin was detected in lily pollen. For ectopic expression of PIPs in the lily pollen protoplasts, we constructed plasmids in which Arabidopsis AtPIP1;1 or AtPIP2;1 was under the control of a strong pollen-specific promoter (maize Zm13). The PCR-amplified DNA fragments were transformed into the pollen protoplasts by electroporation. Between 45 and 60% of protoplasts were successfully transformed. The protoplasts expressing AtPIP2; 1 significantly increased in volume in a hypotonic solution (350 mM mannitol), compared with the vector control. In contrast, the changes in volume of the protoplasts expressing AtPIP1; 1 were similar to that of the vector control. This result suggests that PIP2 induces higher water-flux activity in plant cells, whereas PIP1 does not. Thus, we propose the lily pollen protoplast as a simple and useful experimental system to analyze the function of plant aquaporins.

DOI： 10.5511/plantbiotechnology.11.0922a

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In several plant species, late embryogenesis abundant (LEA) genes are reported as the embryogenesis-related genes that are specifically expressed in embryonic tissues. Several LEA genes have been isolated for carrot somatic embryogenesis. The carrot LEA genes DC8 and ECP63 have very similar nucleotide sequences, and the gene pair is likely to be a nucleotide polymorphism. In several carrot cultivars, plants in which only DC8 was detected (DD genotype), only ECP63 was detected (EE genotype), and both DC8 and ECP63 were detected (DE genotype) have been observed via genomic PCR. Furthermore, distinct frequencies of the genotypes were observed in some carrot cultivars. These results suggested that detection of the nucleotide polymorphism might be useful in defining carrot cultivars. In addition, DC8 and ECP63, which include 14 amino acid insertions/deletions and 21 amino acid substitutions, may have similar effects on somatic embryogenesis, as all genotypes (DD, DE, and EE) were detected in some embryogenic cell lines. Therefore, the nucleotide polymorphism may be a useful candidate polymorphic marker that is easily detectable by PCR. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.scienta.2010.05.001

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.15015/00000323

CiNii Books

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Other Link： http://id.nii.ac.jp/1246/00000323/

Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues. (C) 2008 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.plaphy.2008.02.010

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT CELL & MOLECULAR BIOL  

Carrot (Daucus carota) somatic embryos have been extensively used as an experimental system for studying plant embryogenesis. In maturing zygotic embryos, the phytohormone abscisic acid (ABA) is involved in induction of dormancy and desiccation tolerance. Somatic embryos contain only low levels of endogenous ABA and lack desiccation tolerance and dormancy, but both tolerance and dormancy can be induced by application of exogenous ABA. We analyzed the effects of the optical isomers of ABA, (S)-(+)-2-cis-ABA [(+)-ABA] and (R)-(-)-2-cis-ABA [(-)-ABA], and a racemic mixture of these two isomers (racemic ABA) on the carrot system. Somatic embryos treated with (+)- ABA, (-)ABA, or racemic ABA showed the same levels of growth inhibition and desiccation tolerance. Similarly, each ABA isomer and racemic ABA had the same effect on accumulation of transcripts for ABA- inducible genes. The results indicate that both (+)-ABA and (-)-ABA cause the same amount of activity in these physiological phenomena. Our findings suggest that strict steric requirements of the ABA signaling pathways are absent in carrot somatic embryos, and we propose that commercially available racemic ABA is as effective as the natural isomer (+)-ABA in induction of dormancy and desiccation tolerance in carrot somatic embryos.

DOI： 10.5511/plantbiotechnology.25.457

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Aquaporins facilitate water flux across biomembranes in plant cells. and are involved in various physiological phenomena in several plant tissues. To determine whether aquaporins have physiological roles during seed development, we analyzed the expression of genes encoding plasma membrane intrinsic proteins (PIPs) during seed and fruit development in tomato (Lycopersicon esculentian Mill.). Six genes encoding PIPs were detected in mature tomato seeds by RT-PCR using PCR primers corresponding to conserved transmembrane domains and NPA motifs. The expression of these genes in developing seeds and fruit was analyzed by RT-PCR using primers specific for nine tomato PIPs, including the six PIps detected and an additional three PIPs from the tomato EST database. Increased expression of seven PlPs was detected during the earlier phase of seed development [12-32 days after flowering (DAF)], and the expression of these genes decreased during the later phase (36-56 DAF). Each tomato PIP showed a distinct expression pattern during fruit development. In addition. the water content of the cells was calculated. The seed water content decreased gradually in the earlier phase of seed development (12-32 DAF). and was subsequently maintained at 44-50% from 36 to 56 DAF, whereas the water content of the fruit remained at 90% throughout fruit development. These results suggest that plasma membrane aquaporins play a physiological role during seed and fruit development in tomato. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.plantsci.2006.03.021

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Plant Cell and Molecular Biology  

In carrot (Daucus carota L.) somatic embryos, desiccation tolerance is induced by treatment with abscisic acid (ABA). Six cDNA clones that showed ABA-enhanced expression were isolated from carrot by differential screening with ABA-treated and ABA-untreated somatic embryos, and they were named the CAISE (carrot ABA-induced in somatic embryos) genes. Five of the clones encode late embryogenesis abundant (LEA) proteins, and the other clone encodes a glucose and ribitol dehydrogenase. The expression of the CAISE genes was detected in maturing seeds, embryogenic cells, and ABA-treated somatic embryos in which exhibit desiccation tolerance induced by endogenous or exogenous ABA. These results indicate that ABA-induced desiccation tolerance in carrot somatic embryos may be induced by the LEA proteins and the glucose and ribitol dehydrogenase encoded by these ABA-inducible genes.

DOI： 10.5511/plantbiotechnology.21.309

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER-VERLAG  

LP28, a pollen-specific LEA-like protein identified in Lilium longiflorum purportedly related to the desiccation tolerance of pollen, was localized during male gametogenesis using immuno-clectron microscopy. At premeiotic interphase, LP28 label is absent from the microsporocyte. LP28 label was first detected in the cell wall of the microsporocyte at meiotic prophase I. LP28 gradually increased as the cell wall thickened. In the dyad, after the first meiotic division, LP28 label also appeared in the septum. In the tetrad, after the second meiotic division, LP28 was detected throughout the cell wall, including the septa. Immunolabeling of callose during meiosis indicated that the appearance and localization of LP28 was very similar to that of callose. After the microspores were released from the tetrad by digesting the callosic cell wall, LP28 was not found in the microspores. In bicellular pollen, just after microspore mitosis, LP28 appeared in the generative cell wall, which also consisted of callose. After pollen germination, LP28 also accumulated in the callosic layer of the elongated pollen tube wall and the callose plug. Thus, LP28 colocalized with the callosic cell wall during male gametogenesis. The possible role of LP28 with respect to wall formation during meiosis and pollen development is discussed.

DOI： 10.1007/s00497-002-0142-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

A novel pollen-specific LEA-like protein, LP28, was detected in Lilium longiflorum using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Immunoblot analysis using antiserum raised against LP28 revealed that the protein was not found in somatic tissues or uninucleate microspores, but accumulated gradually in developing pollen following microspore mitosis. Furthermore, LP28 was abundant in germinated pollen after hydration. The cDNA clone corresponding to LP28 encoded a putative protein of 238 amino acids with a calculated molecular mass of 24.2 kDa and a pI of 4.7. The amino acid sequence is highly hydrophilic except for the N-terminal hydrophobic signal peptide. The sequence has similarities with group 3 LEA (late embryogenesis abundant) proteins. Immunocytochemical analyses demonstrated that LP28 was mainly found in cytoplasmic granules of the vegetative cell until pollen maturation, but after hydration it appeared in the elongating pollen tube wall. LP28 might be a unique pollen-specific protein that is transported to the pollen tube wall after germination. Therefore, it is assumed that LP28 plays a role not only in pollen maturation, but also in the growth of the pollen tube, which penetrates the stylar matrix.

DOI： 10.1046/j.1365-3040.2002.00852.x

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：GUSTAV FISCHER VERLAG  

To obtain direct evidence for the involvement of C-ABI3, a carrot (Daucus carota L.) homolog of VP1/ ABI3, seed-specific transcription factor, in the acquisition of desiccation tolerance, we prepared transgenic carrot non-embryogenic cells (NC) in which the C-ABI3 gene was expressed ectopically. Non-transgenic NC, in which expression of C-ABI3 was not detected, did not exhibit desiccation tolerance even after treatment with abscisic acid (ABA). In transgenic NC that expressed C-ABI3, embryo-specific ABA-inducible genes (ECP genes) were expressed upon ABA-treatment. Furthermore, the transgenic NC became desiccation-tolerant upon ABA-treatment, but did not tolerate desiccation without ABA-treatment.

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

We have previously reported that strong desiccation tolerance in carrot somatic embryos can be achieved by treatment with abscisic acid (ABA). In this study, we examined the possibility of long-term preservation of ABA-treated and desiccated somatic embryos. Somatic embryos that had been desiccated after treatment with ABA survived for at least 169 weeks at -25 degrees C. By contrast, somatic embryos that had not been desiccated after treatment with ABA survived for at least 24 weeks at +5 degrees C but died at -25 degrees C.

DOI： 10.1007/s002990050654

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT PHYSIOLOGISTS  

A carrot gene homologous to the ABI3 gene of Arabidopsis was isolated from a carrot somatic embryo cDNA library and designated C-ABI3. The sequence of C-ABI3 was very similar to those of ABI3 of Arabidopsis and VPI of maize in certain conserved regions. The expression of C-ABI3 was detected specifically in embryogenic cells, somatic embryos and developing seeds. Thus, expression of C-ABI3 was limited to tissues that acquired desiccation tolerance in response to endogenous or exogenous abscisic acid (ABA). Endogenous levels of ABA in seeds increased transiently and then desiccation of seeds started. The expression of C-ABI3 in developing seeds was observed prior to the increase in levels of endogenous ABA that was followed by desiccation of seeds. In transgenic mature leaves in which C-ABI3 was ectopically expressed, expression of ECP31, ECP63 and ECP40 was induced by treatment with ABA, which indicates that the expression of ECP genes was controlled by the pathway(s) that involved C-ABI3 and ABA. This suggests that C-ABI3 has the same function as VP1/ABI3 factor in carrot somatic embryos.

DOI： 10.1093/oxfordjournals.pcp.a029319

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A cDNA clone, designated DcDB1, was isolated from a cDNA library prepared from embryogenic cell clusters of carrot (Daucus carota L.) and characterized. The cDNA (1416 bp) encoded for a protein of 392 amino acid residues that contained a conserved chrome domain. The chrome domain is a 37 aa region found in both the Polycomo gene product, which is a repressor of homeotic genes, and a heterochromatin protein 1 of Drosophila. This domain is postulated to function in the binding of proteins to chromatin. Genomic blot hybridization experiments suggested that the number of DcCB1 genes in the carrot genome is low. The level of DcCB1 mRNAs was high in somatic embryos at globular and heart-shaped stages but low in torpedo-shaped somatic embryos. The level of DcCB1 transcripts decreased during the formation of seeds. The existence of both homeo and chrome box genes in plants suggests that regulatory mechanisms of developmental genes in plants may resemble those in Drosophila. (C) 1998 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0167-4781(98)00052-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC PLANT PHYSIOLOGISTS  

Three cDNA clones encoding isoforms of carrot glutamine synthetase (GS) were isolated and used as probes for analysis of the patterns of expression of the genes for GS isoforms during somatic embryogenesis and seed development in carrot. Transcripts corresponding to two of the cDNAs, CGS102 and CGS201, accumulated in both somatic embryos and developing seeds in the same manner. Their levels were high at the early stage of embryogenesis but decreased at the late stage. This pattern of expression is similar to the pattern of changes in GS activity observed during somatic embryogenesis. In contrast, expression of the transcript for another GS isoform detected with CGS103 cDNA was observed at the late stage of seed development and in senesced leaves but not in somatic embryos or young leaves. We also analyzed the levels of the transcripts in somatic embryos that had been cultured in media with either ammonium ions or glutamine as the nitrogen source. The amounts of the CGS102 and CGS201 transcripts fell when glutamine was supplied in the medium. These results indicated that GS activity was regulated at the transcriptional level and that the pattern of expression of the genes for GS during somatic embryogenesis reflected that during zygotic embryogenesis, It is possible that somatic embryogenesis and zygotic embryogenesis have common regulatory systems with respect to nitrogen metabolism.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

An 18 kDa extracellular insoluble protein (EIP18) was found previously in amorphous particles suspended in the culture medium and in the interspaces of cell clusters of carrot (Daucus car-ota L.) callus, as well as in the extracellular spaces of carrot seeds, being located both in the embryo and at the inner edge of the endosperm. We purified EIP18 by washing the amorphous particles with the mixture of Triton X-100, NaCl and ethylenediaminetetraacetic acid (EDTA). We determined several partial amino acid sequences, and then we cloned and sequenced a cDNA for EIP18. EIP18 was found to consist of 133 amino acid residues that included a signal sequence, but it did not contain cysteine, sites for N-linked glycosylation or hydrophobic regions. Since its sequence was found to be homologous to that of inhibitors of cysteine proteinases, namely cystatins, EIP18 was renamed EICC (extracellular insoluble cystatin of carrot). EICC expressed in yeast was also found in an insoluble form in yeast cell walls. EICC prepared from the culture medium of carrot cells inhibited commercial cysteine proteinases and a proteinase extracted from germinating carrot seeds. The expression of the gene for EICC was detected in developing seeds, and the level of its transcript was markedly enhanced upon treatment of somatic embryos with abscisic acid.

DOI： 10.1023/A:1005842719374

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS  

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Language：English   Publisher：Japanese Society of Plant Physiologists  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=184009

Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

It is generally suggested that abscisic acid (ABA) involves in acquisition of desiccation tolerance and dormancy in zygotic embryos. On the other hand, carrot somatic embryos do not synthesize ABA and thus show no desiccation tolerance. We succeeded to induce desiccation tolerance in torpedo-shaped carrot somatic embryos by ABA treatment (1x10-^5 M; 7 days). Generally, it is suggested that VP1/ABI3 factor is important on seed-specific ABA signal transduction. We cloned a homolog of ABI3 gene, C-ABI3, from carrot somatic embryos. The expression of C-ABI3 was detected specifically in developing seeds, somatic embryos and embryogenic cells by Northern blot analysis. These tissues acquire desiccation tolerance by endogenous or exogenous ABA. Here, we made the transgenic plants showing ectopic expression of C-ABI3 in mature leaves or non-embryogenic cells (NC). The expression of some embryo-specific ABA-inducible genes was detected in ABA-treated (1 x 10-^5 M or 1 x 10-^4 M; 8 h or 24 h) mature leaves or NC of the transgenic plants by Northern blot analysis. Furthermore, desiccation tolerance was observed on ABA-treated (1 x 10-^4 M; 14 days) transgenic NC. These all results induce a conclusion that C-ABI3 functions on acquisition of desiccation tolerance through the control of expression of some ABA-inducible genes not only in zygotic embryos but also in somatic embryos of carrot.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC PLANT PHYSIOLOGISTS  

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Language：Japanese   Publisher：The Japanese Society for Chemical Regulation of Plants  

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Language：Japanese   Publisher：植物化学調節学会  

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Language：Japanese   Publisher：植物化学調節学会  

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Event date： 2019.8 - 2019.9

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